The studies outlined in this proposal are aimed at providing a better understanding of Vibrio cholerae virulence determinants which are involved in intestinal colonization. The goal of this project is the molecular analysis of a region of the V. cholerae chromosome which contains a set of four coordinately regulated genes (acf loci, accessory colonization factor) that are involved in the colonization properties of V. cholerae. Insertional mutations in any one of these four genes produces a quantitatively identical colonization defect in suckling mice. The proposed experiments are intended to elucidate the organization, expression, and function of genes specifying the ACF phenotype. The four genes encoding Acf determinants will be isolated and subjected to both DNA sequence analysis and computer aided amino acid similarity search. This region of DNA will be subjected to transposon and deletion mutational analysis in order to: define the limits of each acf gene; locate promoters and controlling elements:; and identify additional genes involved in ACF expression. Acf gene products will be identified, characterized, and localized to specific bacterial compartments with antisera raised against PhoA fusion proteins, purified acf proteins, or synthetic peptides using immunoblot analysis, fluorescence microscopy and colloidal gold labeled antibody immuno-electronmicroscopy. V cholerae strains with defined acf mutations will be constructed in ctxA deletion backgrounds by in vivo marker exchange for use as potential vaccine candidates. An adult rabbit animal model will be assessed for its suitability in characterizing (protective?) host immune responses to Acf determinants. E1 tor strains of V. cholerae (the biotype responsible for the current pandemic) will be characterized with respect to ACF; to date these genes have been characterized only for a classical strain. Knowledge gained from the proposed investigation may provide a basis for the development of efficacious cholera vaccines.